Traditional heterogeneous detection methods for nucleic acid require separation of hybridized and unhybridized probes, while new homogeneous methods eliminate the separation steps, and are more rapid, simple and quantitative. A variety of nucleic acid amplification techniques have been developed and can amplify a specific sequence of nucleic acid to several million copies within 2-3 hours. However, the dominant gel electrophoresis analysis greatly hindered their wide application in clinical diagnostics. Recently, the combination of homogeneous detection with these amplification techniques, especially polymerase chain reaction (PCR), greatly improved nucleic acid based diagnostics. The resulting quantitative real-time PCR assays are becoming increasingly popular.
Current real-time fluorescence PCR assays can be classified into probe format and non-probe format. Probe format assays utilize fluorogenic probes, e.g. 5′-exonuclease (TaqMan™) probes, molecular beacons, fluorescence energy transfer probes, Scorpion probes, light-up probes, etc. Non-probe format assays utilize fluorogenic dyes, e.g. SYBR Green I, to indicate the reaction. The non-probe format, though simple, finds rather limited application due to its inability to discriminate non-specific amplification. In comparison, the probe format with a second recognition step is much more reliable. However, current probes mentioned above are all difficult to design and synthesize, and they are expensive. Another disadvantage of current probes is their limited specificity. Even molecular beacons, which are claimed to be the most specific ones, have to be modified to discriminate single-nucleotide mismatch in some cases.
This invention relates to a new probe that can be a homogeneous and specific probe for nucleic acid detection. This probe is based on a concept different from the current probes. It is simple to design, easy to prepare, inexpensive, extremely specific, and can be combined with any current nucleic acid amplification technique.
Assays with double-stranded probes according to this invention are based on competitive reaction between oligonucleotides rather than direct hybridization as utilized in current probes. This new probe not only can achieve in a much simpler way what the current probes can, but also possesses many advantages over the current probes.